Methods and compositions for inducing monocyte cytotoxicity

ABSTRACT

The present disclosure relates to a new lymphokine molecule, referred to as Monocyte Cytotoxicity Inducing Factor (MCF), and its use as in cancer and other types of therapy. The disclosure further relates to the development of novel Sezary cell hybridomas which secrete MCF and thereby provide a ready source for MCF isolation and purification. 
     Sezary&#39;s Syndrome is a leukemic proliferation of OKT4+ lymphocytes. Sezary cells were isolated by differential centrifugation and fused to CEM.8aza r/ . C, an HGPRTase lacking clone of CEM. The hybrid cells were studied for their ability to produce soluble mediators of human monocyte cytotoxicity. The product of a single clone, FtF3, which bore the surface phenotype of Sezary cells, was characterized. Monocyte cytotoxicity inducing factor was found to be stable at pH 2 for one hour, unlike interferon-gamma, and was found to be more heat stable as well. Moreover, treatment of MCF with antisera to interferons gamma, alpha, or a combination of gamma and alpha failed to neutralize its biologic activity. 
     MCF binds to Matrex Gel Red A. MCF eluted-from this dye-ligand was found to have an apparent molecular weight of 11,500 Daltons by gel filtration and 14,700 Daltons by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 
     A molecular weight of 29,000 daltons was found by use of SDS-PAGE in a second species of MCF produced under serum-free conditions.

This application is a divisional of copending application Ser. No.07/417,162, filed Oct. 4, 1989, now U.S. Pat. No. 5,112,948 and acontinuing application of copending U.S. Ser. No. 06/917,983, filed Oct.10, 1986, now U.S. Pat. No. 4,977,245, issued Dec. 11, 1990.

Reference is hereby made under 35 U.S.C. §120 to copending applicationU.S. Ser. No. 917,983 filed Oct. 10, 1986, incorporated herein byreference.

BACKGROUND OF THE INVENTION

The government may own certain rights in the present invention pursuantto Public Health Services grant NIH R23-CA39441-01.

1. Field of the Invention

The present invention is directed to biological compositions and methodsfor inducing human monocytes to a cytoxic state. More particularly, thepresent invention relates to a soluble factor which induces monocytecytotoxicity and antitumor activity.

2. Description of the Related Art

Immune protection of vertebrates is provided by a dual system thatmaintains two basic defenses against foreign invaders. These twodefenses, termed cellular and humoral immunity, are adaptive and respondspecifically to most foreign substances, although one response generallyis favored. While cellular immunity is particularly effective againstforeign tissue, cancer cells, intracellular viral infections andparasites, the humoral immune response defends primarily against theextracellular phases of bacterial and viral infections. Therefore, thecellular response is directed primarily against invading cells, whilethe humoral response is directed against primarily cell products, suchas toxins. Moreover, whereas cellular immunity is provided by cells ofthe lymphoid system, humoral immunity is provided by proteins calledantibodies that circulate through the fluid compartments of the body.

The dual nature of the immune system is generated from two separatepopulations of morphologically indistinguishable lymphoid cells calledlymphocytes. While one class of lymphocytes, the T-cell lymphocytes,mediates the cellular immune response, the other class of lymphocytes,the B-cells, is responsible for the humoral immune response. Thus, whenthe organism is invaded by a foreign substance, for example an alteredcell (e.g. viral transformed cell or tumor cell), some of the T-cellsthat recognize it are activated and initiate reactions that includebinding to and eliminating the altered cells. On the other hand, whenindividual B-cells are activated, they differentiate to plasma cellsthat secrete specific antibodies directed against substances secreted bythe foreign invader. For a good review of the foregoing, see Hood etal., Immunology, Second Edition, 1984, Benjamin/Cummings PublishingCompany, Inc., Menlo Park, Calif.

While cells of B-lymphocyte lineage have found widespread clinical andindustrial application in the generation of monoclonal antibodies, cellsof T-lymphocyte lineage have proved of interest in part due to thenumerous soluble factors they secrete. In the biologic system, T-cellfactors play an important role in modulating and activating variousimmune functions. Isolation and characterization of various T-cellfactors has been the goal of many clinical research endeavors attemptingto identify those factors which might be useful in treating a number ofdisease states, for example, in the treatment of tumor cells and viralinfectious states. Of particular interest has been the recentcharacterization of a factor termed T-cell growth factor, or interleukinII, which is produced and secreted by effector T_(A) cells. (See U.S.Pat. Nos. 4,401,756; 4,404,208; 4,407,945; and 4,473,642). When T_(C)cells are stimulated by interleukin II, they undergo an effector phaseand are stimulated to mature into killer T-cells which are capable ofidentifying and eliminating various target cells. As demonstrated by theabove patents, interleukin II has become an important pharmaceuticalagent in the treatment of various disease states.

Optimism spurred by the preliminary success of interleukin II have leadresearchers on a quest to identify other immune-mediating factors havingpotential clinical applicability. However, this search has generallybeen hampered by the existence of numerous factors secreted by the sameor similar cell types. Moreover, confusion often results from thegeneral overlapping nature of the factor activities and often times froma lack of currently available test systems for identifying individualfactor activities. Without highly sensitive test systems for identifyingindividual factor activities, the existence of a particular factorcannot be readily distinguished from other factor activities.

Recently, interest has been shown in identifying soluble factors whichserve to stimulate human monocyte cytotoxicity. Monocytes are aphagocyte of the blood which, along with macrophages andpolymorphonuclear leukocytes, bind and ingest foreign substances oftenprior to an antibody response. "Activated" monocytes have recently beenshown to exert an antitumor activity. For example, Fischer et al. (Cell.Immunol., 58:426-435 (1981)) disclose that human peripherial bloodmonocytes can reproducably lyse a variety of tumor cells. More recently,researchers have disclosed various factors thought to play a role inmonocyte activation. For example, Kleinerman et al. (Cancer Res.,45:2058-2064 (1985)), discusses the activation of human blood monocytesby incubation with concanavalin A-stimulated lymphokine(macrophage-activating factor (MAF)), lipopolysaccharide endotoxin, andhuman recombinant gamma interferon. It was reported that gammainterferon, in the presence of endotoxin, was capable of activatingmonocyte tumoricidal activity. Moreover, MAF treatment exhibited asimilar effect.

Other monocyte cytotoxicity promoting factors have been identified aswell. For example, Le et al. (J. Immunol., 131:2821-2826, 1983) hasreported a T-cell hybridoma line capable of producing a macrophageactivating factor with the ability to activate human blood monocytes toshow enhanced cytotoxicity against a human colon adenocarcinoma line.However, this activity was found to be neutralized with specificantiserum to purified human interferon-gamma. These authors concludedthat this MAF was in fact interferon-gamma.

More recently, Jones and Clouse (Immunobiol., 167: Abstract No. 365(1984)) reported the use of lymphocytes from patients with Sezary'ssyndrome in the production of a human T-cell hybridoma line which iscapable of producing a factor which stimulates monocyte antitumorcytotoxicity. In contrast to the factor identified by Le et al., thefactor reported in the Jones and Clouse publication was not inhibited byantibodies having specificity for interferon-gamma. Although the Joneand Clouse reference did observe that two molecular weight specieshaving the biologic activity was observable, the methodology used toidentify this particular activity was not identified. Moreover, themethodology for identifying and isolating Sezary/T-cell hybridomas whichsecrete the particular factor was not disclosed. Clearly, not allSezary/T-cell hybridomas are capable of producing monocyte stimulatoryfactors (See, e.g., Grillot-Courvalin et al., "Helper T-HybridomaProduced By Fusion With Sezary Cells," in: T-Cell Hybridomas, ed. by M.J. Taussig, CRC Press, Inc., Boca Raton, Fla., 1985).

It is apparent from the foregoing references that, not only are therenumerous factors potentially involved in the stimulation of monocytecytotoxicity, but additionally that these factor activities may beindistinguishable in previously available assays for detecting variouscytotoxic actions.

Accordingly, the present invention is directed to methods foraccomplishing the isolation of particular discrete soluble factors whichexhibit monocyte cytotoxicity inducing activity. Moreover, the presentdisclosure is directed to a detailed characterization of these factorsand to the preparation of T-cell hybridoma lines which produce thesefactors in vitro and thereby provide a ready source for isolating thefactors. In that the novel factors of the present invention demonstratea surprising ability to elicit an antitumor response by monocytes invitro, similar to that possessed by interleukin II for lymphoid cells,it is believed that these factors will provide an important new additionto the antineoplastic armament of medical science.

SUMMARY OF THE INVENTION

The present invention is directed to a composition which includessubstantially purified human monocyte cytotoxicity inducing factor, alsoreferred to herein as MCF. The composition includes a substantiallypurified soluble factor capable of inducing human monocytes to acytotoxic state, the factor having a molecular weight of between aboutis 18,100 and about 14,700 Daltons when determined by polyacrylamide gelelectrophoresis under the conditions as described herein. It will beappreciated that under differing electrophoresis conditions, thismolecular weight may vary.

When subjected to gel filtration chromatography, the factor has anapparent molecular weight of approximately 11,500 Daltons whenchromatographed under the conditions described herein. Again, as withgel electrophoresis, it will be appreciated that gel filtrationchromatographydoes not provide an exact molecular weight determination,rather such molecular weight determinations appear as a broad elution ofmonocyte cytotoxicity inducing activity, wherein the peak activityelutes from such columns at a position which corresponds toapproximately 11,500 Daltons. However, this molecular weight may varywith variations in conditions, for example, running buffer, exclusionlimit, column size and the particular gel filtration methodology whichis utilized.

The present invention is additionally directed to a substantiallypurified soluble factor capable of inducing human monocytes to acytotoxic state wherein the factor exhibits a molecular weight of about29 kilodaltons by gel electrophoresis chromatography when the factor isobtained from cells grown in serum-free media, and a molecular weight atbetween about 78 and 63 kilodaltons, essentially co-eluting with bovineserum albumin by gel filtration chromatography, when grown in mediacontaining serum. In that bovine serum albumin has a molecular weight ofapproximately 68,000 Daltons, it is likely the case that this highermolecular weight species of MCF has an affinity for serum albumin. Thiswould explain why, when obtained from cells grown in the presence ofserum albumin, the higher molecular weight factor exhibits a molecularweight essentially indistinguishable from that of serum albumin.

A composition which includes one or both of the above factors may befurther characterized by physicochemical and biologic characteristics.For example, the composition and factor(s) retains biologic stabilityfollowing treatment at pH 2 for one hour. The term biologic stability,as used herein, is defined as the retention of substantial biologicactivity following the indicated treatment as defined by the presentdisclosure. The composition similarly retains biologic stabilityfollowing treatment at 60 degrees Centigrade for one hour, and furtherretains biologic activity in the presence of antiserum tointerferon-gamma, interferon-alpha, or a combination of antisera tointerferon-alpha and gamma. MCF activated monocytes retain biologicalactivity in the presence of antiserum to Leu-11b plus complementindicating that the factor does not appear to activate NK cells, andfollowing treatment by the enzymes RNase, DNase and trypsin. Biologicactivity is reduced following treatment by the enzyme chymotrypsin. MCFproduced under serum-free conditions demonstrates charge heterogeneity,with three species having isoelectric points at 2.7, 5.6, and 6.7,respectively.

The present invention is additionally directed to a continuous cell linewhich produces a factor as defined by the foregoing characteristics. Inparticular, a continuous cell line is provided which produces a factorcapable of inducing human monocytes to a cytotoxic state, wherein thecell line is produced by a process which includes the steps ofimmortalizing human T-cells to produce continuous cell clones;identifying a clone which produces the factor; and culturing the cloneto provide the continuous cell line.

The first step of immortalizing human T-cells to produce continuous cellclones is generally defined as providing human T-cells in a mannerwhereby they may be cultured continuously for an indefinite period. Themost convenient manner for providing such continuous cell clones isthrough the development of a T-cell hybridoma. T-cell hybridomas aregenerally well known in the art and may be generated by a variety ofwell known methods. In general, such methods include fusing humanT-cells with a second cell population which is sensitive to growth in aselective media and culturing the fused cells in the selective media toproduce the continuous cell clones. Cells may be fused in numerous ways,for example, through the use of polyethylene glycol or Sendai virus.

In a preferred embodiment, the second cell population is a T-celllymphoma population which has been selected for growth in 8-azaguanine.By selecting for T-cell lymphomas capable of growth in 8-azaguanine, acell population sensitive to growth in HAT selective media is obtained.Those of skill in the art will recognize that other cell lines havingother selective criteria may be utilized for fusion with human T-cellsto provide the continuous cell clones. For example, additional T-celllymphoma subtypes could be used to clone other subclasses of humanT-lymphocytes. Moreover, drug sensitivities and other selective criteriacan be generated using other approaches including, 6-thioguanine,oubaine or oncogenic transformation. Additionally, inter-species hybridscan be generated to allow for chromosomal localization.

However, fusion is not the only means of achieving immortalized humanT-cells. For example, certain human T-cells are responsive to T-cellgrowth factor and may be immortalized by continuous culturing in thepresence of certain human T-cells, for example, certain neoplasticT-cell lines are capable of continuous growth in cell culture as arecertain transformed (e.g. virus transformed) T-cell lines. While mostT-lymphotrophic viruses are toxic, it is known that HTLV-1, as well asportions of the EB virus gerome, commonly utilized in B-lymphocytetransformation, can be used to transform and thereby immortalize,T-lymphocytes. All such continuously growing T-cells, and methods ofproviding continuously growing T-cells, are included within the scope ofthe present invention.

After obtaining the immortalized human T-cells in the form of continuouscell clones, a clone is identified which produces the monocytecytotoxicity inducing factor. The crux of the successful practice of thepresent invention relies on the ability to identify clones which producethis factor rather than the numerous other immune regulatory andstimulatory factors known in the art. It is now believed that manyhundreds of peptides, whose functions are unknown, are secreted byvarious activated T-cells. (see, e.g., Zurawski et al. (1986) Science,232: 772-775). Moreover, depending on the particular T-cell which isimmortalized, the number of clones positive for factor production may bequite low. Therefore, the assay must be not only highly specific for thepresent factor, but must be quite sensitive to the presence of smallamounts of the factor, in order to successfully practice the presentinvention. Accordingly, the present disclosure is directed to an assayparticularly adapted to identification of the present factor.

The final step of the present process is simply culturing the identifiedclone to produce the cell line. Where the immortalized human T-cell isachieved through hybridoma development, culturing will include simplyculturing in an acceptable media. However, where the immortalized cellline does not involve cell fusion and instead requires the presence of agrowth maintaining factor such as T-cell growth factor, culturing willrequire the inclusion of the particular growth factor.

In that it is believed that the factor of the present invention issecreted by a very small proportion of T-cells in general, it is apreferred embodiment of the present invention to employ Sezary cells asthe T-cells to be immortalized. This is because it has been determinedthat a relatively large proportion of Sezary cells do in fact producethe present factor. However, in that it appears clear that not allSezary cells produce the factor, the selection step is still required inorder to identify clones producing the factor. However, if Sezary cellsare unavailable, and one does not desire to screen the large number ofclones which must necessarily be screened where T-cells in general areutilized, one may desire to use effector T-cells. It has been determinedthat a population of effector T-cells include a larger proportion offactor positive cells than do T-cells in general.

In a very general sense, the method of identifying a clone whichproduces the factor includes the steps of stimulating the clone with aT-cell mitogen to release lymphokines; culturing human monocytestogether with appropriate target cells, for example, human cancer cells,in the presence of the released lymphokines; and detecting target celllysis, wherein such lysis is indicative of the presence of humanmonocyte cytotoxicity inducing factor in the released lymphokines. Asused herein, and as appreciated in the art, lymphokines is a genericterm directed to any molecule having biologic activity for modulatingthe immune system. A T-cell mitogen, as will be appreciated by those ofskill in the art, is a molecule or a compound having the ability tostimulate the release of lymphokines from T-cells. In a preferred aspectof the present invention, the T-cell mitogen used is phytohemagglutin,commonly referred to as PHA. However, concanavalin A and other T-cellmitogens known to the art may be successfully utilized.

The second step of culturing human monocytes together with appropriatetarget cells in the presence of released lymphokines allows for thespecific induction of monocyte cytotoxicity inducing factor where suchfactor is present in the released lymphokines. As noted previously, thisparticular step is quite important to the successful practice of thepresent invention and is disclosed in detail in connection with thedisclosure of a preferred embodiment in a later section. Of course, todemonstrate that the present factor is indeed effective against humantarget cells, an appropriate human target cell is preferred. Usefultarget cells have been identified as the human myeloid leukemic cellline, K562 and also HL60, L5178Y and TU5 cells. This group of tumortargets includes both NK-sensitive and NK-resistant cells. However it isbelieved that numerous additional cell types may be employed, forexample, melanoma, lung carcinoma and bladder cell tumors.

The final step of detecting target cell lysis is conveniently performedthrough the use of a radioisotope which is maintained extra orintracellularly when the target cell is in a non-lysed condition, andwherein the radioisotope is released into the surrounding media when thetarget cell is lysed. However, it will be appreciated that additionalmethods known in the art, and disclosed herein, may be used.

The present invention is additionally directed to a method forgenerating substantially purified monocyte cytotoxicity inducing factorwhich includes the steps of:

(a) stimulating a continuous cell line which produces the monocytecytotoxicity inducing factor with T-cell mitogen to release the factorinto the culture supernatant;

(b) subjecting the supernatant to gel filtration chromatography;

(c) assaying chromatography fractions for human monocyte cytotoxicityinducing activity; and

(d) collecting those fractions which exhibit the activity.

Practicing the method to this extent will provide a factor containingcomposition which is substantially purified with respect to biologicactivity as defined by the disclosed assay. However, it is believed thatsufficient biologic purity may be obtained by simply passing the culturesupernatant over a Matrex Gel Red A (Amicon) column in a lowsalt-containing buffer to bind the factor to the Matrex gel, washing thecolumn to remove non-binding material, and eluting the bound fractionfrom the column with a high salt-containing buffer. Alternatively, toachieve an even more purified factor preparation, one may combine thegel filtration chromatography procedure with the Matrex gel red A columnprocedure. Even further purification may be obtained by combining eitheror both of the foregoing procedures with a polyacrylamide gelelectrophoretic separation. It has additionally been determined thatisolectric focusing techniques, which are generally well known in theart, provide for purification of MCF.

In that it is believed that the monocyte cytotoxicity inducing factor ofthe present invention will be clinically applicable in a manner similarto that identified for interleukin II and the various interferons,methods are additionally disclosed for clinical utilization andtreatment of disease states using MCF. In addition to clinicalapplicability with respect to tumor treatment, as indicated bydemonstrated in vitro efficacy in stimulating monocyte antitumoractivity, it is believed that pharmaceutical compositions of the presentinvention will be useful in the treatment of infectious diseases,particularly those infectious diseases wherein the causative organismsreside in mononuclear phagocytes, for example, tuberculosis andleshmaniasis.

Additionally, it is believed that compositions included by the presentinvention will find diagnostic utility. Antiserum specific for MCF wouldbe of value in determining blood levels of MCF, as well as documentingthe ability of patients' mononuclear cells to produce MCF. Since MCF isproduced by T-lymphocytes, and in particular, neoplastic T-lymphocytes,MCF will likely serve as a marker for diagnosis and/or evaluation ofT-cell malignancies.

With respect to therapeutic utilization of MCF, one treatment protocolwould include the ex vivo activation of a patient's mononuclear cellsfor reinfusion into the patients in a manner analogous to LAK cells asdescribed by Rosenberg et al. (J. Natl. Cancer Inst., 75:595, 1985, andN. Eng. J. Med., 313:1485, 1985). For direct delivery of MCF to tissuemacrophages, it is contemplated that MCF may be given by directtransfusion, as well as being encapsulated in liposomes. Such techniqueshave recently been found to increase the efficacy and significantlyprolong the half-life of related low molecular weight mediators.Liposome encapsulation can be accomplished in a number of manners, forexample, as described by Fidler et al. (1976) Cancer Res., 36:3608,incorporated herein by reference.

Additionally, combination therapy employing MCF in combination withinterleukin II, interferon, tumor necrosis factor and cytoxan, arecontemplated. These additional agents may be obtained and employed in amanner known in the art as further disclosed herein.

It is further believed that pharmaceutical compositions which includeMCF will find utility in direct infusion treatment in a manner similarto that utilized for interferon treatment. It is believed that dosagedetermination, as well as proper infusion techniques, is well within theskill of the art as exemplified by Goldstein et al. (Cancer Res.,46:4315-4329, 1986), incorporated herein by reference. Moreover, in thatinterferons have found applicability in the treatment of variousinfectious disease states as noted above, it is believed that suchutility will be applicable to MCF as well, for example, when employed assuggested by Nathan et al. (J. Exp. Med., 160:600-605, 1984),incorporated herein by reference.

Diagnostic procedures, utilizing antibodies specific for MCF, may beemployed in a manner similar to the current clinical test for acquiredimmune deficiency syndrome. Most conveniently, this would include astandard enzyme linked immunosorbent assay (ELISA), well known to thoseskilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Hybrid cells were adjusted to 1×10⁶ /ml and stimulated with 8ug/ml PHA(+). PHA was added back to control (-) cultures, and bothsupernatants harvested by centrifugation. Monocyte cytotoxicity wasmeasured as described herein with the exception that the monocytemonolayer was incubated overnight to allow for decay of NK cellactivity. The monolayer was incubated for a second 24 hr period withdilutions of MCF containing supernatants. Dilutions represented are 1/4in a total test volume of 0.2 ml. The supernatant was washed away andthe ¹¹¹ In-Ox-labeled targets, K562 were added (E:T,30:1). Microtiterplates were centrifuged 16 hrs later, and supernatants collected andcounted. % specific release was calculated as described herein.

FIG. 2. 7.2×10³ U of MCF in 28 ml was applied to a 2.5×60 cm column ofBio-Gel P 100 equilibrated with PBS pH 7.4. The column was eluted at 1.6ml/min and 7.5 ml fractions were collected. (.--.) MCF units/ml; (x)represent the molecular weight standards: ribonuclease A 13,700,chymotrypsinogen A 25,000, ovalbumin 45,000, and aldolase 158,000.Molecular weight determinations were calculated using Curvfit program(Interactive Microware, Inc. State College, Pa.) on an Apple II+ system.

FIG. 3. 4 ml FtF3 supernatant was passed twice over 2 ml resin bedvolume and eluted with a salt gradient. 10.5 and 31.3% activity could beeluted with 1M NaCl on Blue A and Red A, respectively. Biologic activitywas not recovered using Orange A, Green A, or Blue B.

FIG. 4. 20 U of MCF/290 ug protein in 0.2 ml was mixed with samplebuffer, heated, and centrifuged as described. 100 ul was loaded intoeach of 4 lanes of a 1.5 mm thick 10% polyacrylamide-SDS gel. Afterrunning at 200 V for 4 hrs, the gel was cut into 1 cm slices, crushed,eluted, and bioassayed. Companion lanes were stained by both CoomassieBlue and silver method. Molecular weight standards used werephosphorylase beta (94,000), bovine serum albumin (67,000), ovalbumin(43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor(20,000), and α-lactalbumin (14,400). Molecular weight determinationswere made using Ferguson plot analysis.

FIG. 5. FtF3 cells were incubated 4 h with various doses of actinomycinD. Cells were washed and stimulated with PHA for 24 h before harvestingsupernatants by centrifugation, extensive dialysis, and bioassay. ( )One set of each dose of inhibitor-treated cells was labelled with ³H-uridine, stimulated, lysed and harvested 24 h later, and counted byliquid scintillation.(□) Viability of FtF3 at 24 h was greater than 80%at all doses tested.

FIG. 6. FtF3 cells were incubated 4 h with various doses ofcycloheximide. Cells were washed and stimulated with PHA for 24 h beforeharvesting supernatants by centrifugation, extensive dialysis, andbioassay.( ) One set of each dose of inhibitor-treated cells waslabelled with ³ H-amino acids, stimulated, TCA-precipitated 24 h later,and counted by liquid scintillation.(□) Viability of FtF3 at 24 h wasgreater than 80% at all doses tested.

FIG. 7. FtF3 cells were incubated 4 h with various doses of puromycin.Cells were washed and resuspended either with ( ) or without (□) PHA,and incubated 24 h before harvesting supernatants by centrifugation,extensive dialysis, and bioassay. Viability of FtF3 at 24 h was greaterthan 80% at all doses tested.

FIG. 8A. Biological activity and 15% SDS-PAGE. (10) 5 Low molecularweight standards (BRL, Gaithersburg, Md., U.S.A.) used were ovalbumin,(43K), alpha chymotrypsinogen (25.7K), beta-lactoglobulin (18.4K),lysozyme (14.3K), bovine trypsin inhibitor (6.2K), and insulin A and Bchain (2.3K and 3.4K).

FIG. 8B. 15% SDS-PAGE: Western blot. Solubilization buffer (A); MCFpurified over Matrex Gel Red A (B); low molecular weight standards, BRL(C): stained with colloidal gold (Aurodye).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Introduction

Concepts of monocyte activation for cytotoxicity have undergone a changefrom the "all or none" paradigm to the more precise concept of adiscrete stepwise process. Our present understanding of monocyteactivation has been addressed by Cohn (Jrnl Immunol., 121:813-816,1978). Moreover it is generally recognized that activation is regulatedby products of T-lymphocytes. Soluble mediators of mononuclear phagocyteactivation have recently been reviewed by Nathan et al., supra.

From these studies and earlier reports, interferon gamma would appear tobe the most important if not the only regulator of cytotoxicity againstmicroorganisms and has recently been identified as a migrationinhibitory factor. However, other studies have suggested that colonystimulating factor (CSF I), and the interferons alpha and beta, may haveroles in regulating secretory or proliferative functions. The presentinvention is directed to the perhaps surprising discovery that anadditional factor, termed monocyte cytotoxicity inducing factor (MCF) bythe present inventors also plays a role. In light of the presentfinding, it is likely that additional factors will be identified throughthe development of the individual assays particularly suited toidentification of factor activities.

The MCF disclosed by the present invention is characterized biologicallyby an ability to stimulate monocytes to a cytotoxic antitumor state.While the precise function of MCF within the biologic system is unknown,it is believed that it might play a role in affording specificactivation of the cellular immune system. MCF appears to cause anincrease in the synthesis and release of IL 1 by monocytes, which mayact to either perpetuate the cytolytic state, to directly lysesusceptible tumor targets, or to induce cytostasis. MCF has no TNF,m-CSF, or IFN-like anti-viral activity. Similarly MCF has no activitywhen tested in the IL 1, IL 2, or m-CSF assays. Moreoever, MCF was notdirectly cytotoxic for the targets K562 or L929, indicating that MCF hasno lymphotoxin-like activity of its own. The factor is characterizableas having two molecular weight forms, one about 29,000 Daltons, and alower molecular weight form of between 11,500 and 18,100 Daltons,depending on the particular molecular weight determination methodutilized. It is emphasized that these two molecular weight forms exhibitindistinguishable biologic activity in terms of monocyte activation.Therefore, the term MCF is meant to relate to either of these twomolecular weight forms individually or in combination.

Sensitivity of MCF to the enzymes and metabolic inhibitors describedherein are consistent with a conclusion that MCF is a peptide. Forexample, responses to metabolic inhibitors (actinomycin-D, puromycin,and cycloheximide), suggest that transcription of message is necessaryand that messenger RNA for MCF does not persist. Because treatment ofnon-lectin-treated cells with puromycin did lead to production of MCF,and because total suppression of MCF could not be achieved withcycloheximide and puromycin, these data are consistent with eitherregulation of transcription by a repressor protein or its product, or atthe level of translation, by stabilization of message.

MCF is most conveniently isolated through the preparation of a T-cellhybridoma employing Sezary cells isolated from an individual havingSezary's syndrome. Sezary's syndrome is characterized by a proliferationof leukemic helper T-cells, and is discussed in some detail by Broder etal. (J. Clin. Invest., 58:1297-1306, 1976). Sezary's cells circulate inthe peripheral blood and are known to provide T-cell help forimmunoglobulin production. These cells are identified by theircharacteristic cerebriform nuclei, PAS positive vacuoles, andadditionally by their surface marker phenotype (OKT3+, OKT4+, OKT8-,OKIal-(+)). Helper function has been defined by the ability of Sezarycells to stimulate polyclonal IgG production by B lymphocytes and bytheir production of MIF. Although lymphokine production does notnecessarily segregate exclusively to any one subclass of T cells,Sezary's cells most probably represent a homogeneous clonal expressionof one particular subclass of human T-lymphocytes which can be studiedfor production of lymphokine mediators uncontaminated by other T-cells.

The use of Sezary cells, as noted above, is not crucial to the practiceof the present invention in that it is believed that MCF production is afeature common to lymphocyte populations in general. However, not allcells of a particular T-cell population produce MCF. Thus, where ageneral T-cell population is utilized, only a very small percentage ofcells are likely to produce MCF. In the case of Sezary's cells, itappears as though MCF production is a more generalized phenomenon.Therefore, Sezary's cells are preferred in that T-cell hybridomasproduced from Sezary's cells provide clones wherein there is a muchgreater likelihood that any one particular clone would produce MCF.Conversely, it is likely that when general T-cell populations areemployed for hybridoma production, a substantial number of clones willlikely have to be screened before a positive clone is identified.However, where Sezary's cells are not available, one may employ helperT-cells, in that it appears that helper T-cell populations contain asufficient percentage of positive cells to avoid the exhaustivescreening which would be necessitated by employing general T-celllymphocyte populations.

Recently, strong evidence has been presented that interferon-gamma isthe major mediator of macrophage/monocyte activation, and may beidentical with the lymphokine macrophage activation factor (MAF).Interferon-gamma has also been described as the mediator of inhibitionof mononuclear phagocyte migration and hence may be migration inhibitionfactor (MIF). However, it is known that activated Factor B of thealternative complement pathway (Bb), plasminogen activator (PA), andother products of monocytes will inhibit migration. Nathan et al.,supra, have recently reviewed the range of mediators of mononuclearphagocyte activation.

The production of lymphokines by malignant T-cells is important inunderstanding host defense in conditions with chronic courses such asSezary's syndrome and may reflect production of such mediators bynon-malignant counterparts in the normal host. In developing the presentinvention, attempts were made to expand Sezary cells with interleukin II(IL-2), but these attempts were unsuccessful. Similar results have beenreported by other groups, and have been attributed to the lack of IL-2receptor expression or proliferation of Sezary cells by non-IL-2dependent means. Clearly, as additional cell lines are screened for MCFproduction, cells will be identified which may be immortalized throughthe inclusion of growth factors in the cell growth medium, thus avoidingthe need for hybridoma development.

Previously, hybridization has been utilized to perpetuate subclasses ofmurine T-cells and this technique was applied to Sezary's cells for thepresent invention. Other groups have employed Sezary cell hybridomas tostudy BCGF (B-cell growth factor) production by Sezary's cells. In thepresent invention, six cell lines were obtained by hybridization and allwere capable of inducing human blood monocytes to become cytotoxic forthe myeloid leukemia target, K562. For further study, one hybrid, FtF3,was selected whose phenotype (OKT3+, 4+, 8-, OKII1-, sIgG-) wasidentical to the parent Sezary's cell. It is of interest that all linesproduced a lymphokine capable of inducing monocyte cytotoxicity (MCF).This probably results from positive selection of fusion partners sincesimilar hybridization experience using whole murine T-cell populationshave given rise to much smaller percentages of clones with MAF-likefunction.

Supernatants from PHA-stimulated FtF3 induced human mononuclearcytotoxicity but contained no detectable-interferon. Antisera to nativeinterferon-gamma when added in excess to MCF having no antiviralactivity produced only a 10-15% decrease in specific lysis of the targetK562. Moreover, when formal titrations of the anti-IFN-gamma antiserawere carried out using the constant antibody method previously describedas being optimal for the detection of cross-reactivity with IFN-gamma,no neutralization titer could be determined. In contrast, treatment withthe antisera to interferon-alpha caused a small increase in specificrelease. Addition of both antisera together produced no change inspecific lysis. Both antisera were produced against partially purifiednative human interferons. In the case of the antisera tointerferon-gamma, some cross-reactivity may be detected in that anMCF-like molecule could have been present in the preparation used toraise the antisera which was previously not recognized.

Recently, Schreiber has described the development of a series ofmonoclonal antibodies which are capable of differentially blockingantiviral or MAF activity of interferon-gamma, respectively. Inparticular, monoclonal antibodies directed against the C-terminus ofrecombinant interferon-gamma were reported to block MAF but not theantiviral activity of the recombinant interferon-gamma, while theantibodies to the N-terminus of recombinant interferon-gamma blockedantiviral but not MAF activity. Therefore, these findings could beconsistant with an interpretation that MCF was an altered interferonspecies produced by a T-cell leukemia-derived hybridoma. However, thefact that MCF has recently been determined by the present inventors tobe produced by normal effector T-cells, appears to rebut this theory.

These issues were further approached by direct comparison of thephysicochemical properties of MCF and native interferon-gamma. Unlikeinterferon-gamma, MCF was stable at pH 2 but was partially inactivatedat pH 8 and was much more stable than interferon-gamma. IFN- wasinactivated by trypsin, in contrast to the effect of this enzyme on MCF.These findings would support the concept that MCF is not an alteredinterferon-gamma which has lost antiviral, but not MAF activity, becauseinterferon-gamma lost both MCF and antiviral activity with both low pHand heat treatment.

MCF is a lymphokine distinct from TNF, IL 1, IL 2, m-CSF or IFN. MCF hasno activity when tested in assays for IL 1, IL 2 or m-CSF. MCF had noTNF, m-CSF, or IFN-like antiviral biological activity when compared torTNF, purified human m-CSF, IFN gamma or alpha/beta, respectively. Inaddition, MCF had no cross reactivity with m-CSF in the radioimmunoassayfor m-CSF.

Gel filtration chromatography of concentrated supernatants from cellsraised in the presence of serum, revealed two molecular weight species,one with a molecular weight of 64,000 daltons which co-eluted with themajor protein peak, bovine serum albumin, and a second with a molecularweight of 11,500 daltons. Although yields from our Bio-Gel P-100 columnhave been low, these values differ from the molecular weight of 50,000reported for native human interferon-gamma when chromatographed undersimilar conditions. When such cells were cultured in serum-free media,the higher molecular weight species migrates at about 29 kD upon SDS gelelectrophoresis

MCF was found to bind to Matrex Gel Red A (procionred agarose; Amicon)and eluted with 1-N NaCl. Similar results have been reported for nativeinterferon-gamma. MCF eluted from Matrex Gel Red A was electrophoresedunder reducing conditions on a 15% SDS-PAGE gel. Two molecular weightspecies were identified. The first had a molecular weight between 25,000to 28,000 daltons when produced in serum-free media and the second had amolecular weight of about 14,700 Daltons. The specific activity was 610MCF units/mg protein and 1350 MCF units/mg protein, respectively. Yieldsfrom this procedure have been excellent With recoveries greater than90%, despite treatment with SDS.

Electrophoresis under reducing conditions followed by dialysis to removeSDS has resulted in each case in a diminution of biologic activityassociated with the higher molecular weight form and greater recovery ofthe lower molecular weight form.

The following methods describe the preferred embodiment employing Sezarycells from two patients identified as having the syndrome. To achieve animmortalized Sezary cell line in the form of a Sezary/T-cell hybridoma,Sezary cells were fused with a T-cell lymphoma line conditioned in amanner to be sensitive to HAT selective media. The resulting hybridomaswere found to produce MCF and were capable of being continuouslycultivated. Culture supernatants were found to contain sufficientquantities of the MCF to allow for substantial purification through theutilization of techniques described herein.

EXAMPLES 1. Isolation of Sezary's cells

Mononuclear cells were isolated from 60 ml whole blood from a patientpreviously known to have Sezary's syndrome. Mononuclear cells wereisolated by centrifugation over Ficoll-Hypaque (Pharmacia, Piscataway,N.J.). 7.5 ×10⁷ cells having a phenotype of 87% OKT 3+, 77% OKT 4+, and12% OKT 8+ were recovered from the F/P centrifugation and applied to a35 ml gradient of percoll (Pharmacia) according to the method ofGemlig-Meyling and Waldman (J. Immunol. Method., 33:1, 1980,incorporated herein by reference). Briefly, the gradients were made bymixing 168 ml Percoll with 144 ml 2x PBS, and centrifuged for 40 min at21,000×g in a Beckman Model 71 centrifuge With a SS-34 fixed anglerotor. The cells were suspended in 5 ml of Hank's balanced salt solution(HBSS), layered gently on the gradient, centrifuged at 100×g for 20 minat 20° C. in a swinging bucket rotor. Standard density marker beads(Pharmacia) were loaded on a companion gradient.

Three distinct "bands" were recovered from Percoll. At the interfacebetween the Percoll and sample were 1.5×10⁶ cells, the majority of whichwere polymorphonuclear leukocytes, and were discarded. Band I, 2 cmbelow the interface contained 1.9×10⁷ cells which when studied by flowmicrofluorometry (FMF) were 90.2% OKT 3+, 80% OKT 4+, and 47% OKT 8+,and were morphologically small lymphocytes. Band II, located 5 cm belowthe interface contained 7.8×10⁶ cells, which were 97% OKT 3+, 96% OKT4+, and 17% OKT 8+. Microscopically, these cells were larger and hadcerebriform nuclei. 38% of the total cells applied to the gradient wererecovered.

Cells isolated in Band II were washed and resuspended at 5×10⁵ cells/mlin RPMI 1640 with 10% fetal calf serum (FCS). Aliquots of these cellswere cultured with 10 units/ml final concentration of interleukin IIfrom the Gibbon ape cell line NLA-144. These cells failed to divide orincorporate 3H-Tdr after 72 hrs of culture thus demonstrating a lack ofIL-2 responsiveness.

2. Preparation of 8-azaguanine Resistant T-lymphocytes

Since these particular Sezary's cells were not capable of indefinitesustained growth in tissue culture, it was decided to prepare acontinuous T-cell hybridoma line by fusing the Sezary's cells with aT-lymphocyte population which has been conditioned to grow continuouslyin tissue culture. To achieve hybrid selectability, it was firstnecessary to select a cell population which was sensitive to HATselection medium. This was accomplished through the preparation of an8-azaguanine-resistant T-lymphocyte population.

It has been determined that certain commercially available T-lymphocytecell lines are to be preferred over others. CCRF-CEM is a cell lineobtainable from the ATCC which, due to its apparent genetic stability,is to be preferred in generating hybridomas of the present invention.Two additional cell lines, MOLT and Jurkat, have been found to begenetically unstable upon drug selection and therefore unsuitable.However, the techniques of the present invention are applicable to anygenetically stable T-cell lymphocyte or other cell type which can form astable fusion product with T-cells and which is capable of continuousgrowth in culture. Determination of the foregoing criteria is within theskill of the art and the scope of the present disclosure is not limitedto the preferred embodiment employing CCRF-CEM.

To generate 8-azaguanine resistant CCRF-CEM, cells were obtained fromthe American Type Culture Collection and grown in RPMI 1640 with 10%FCS. CEM was grown in increasing concentrations of 8-azaguanineprimarily according to the method of Okada (Proc. Natl. Acad. Sci.,78:7717, 1981, incorporated herein by reference). However, beginningwith a concentration of 2 uM, the dose was doubled every 2 days until adose of 16 uM was reached at which time the dose was doubled every 10days until a dose of 100 uM was reached. After 6 weeks the cells wererecloned and tested for their ability to grow in HAT-containing media(hypoxanthine, aminopterin, and thymidine), and subjected to flowmicrofluorometry.

3. Generation of T-cell Hybridomas

T-cell hybridomas were formed between Sezary cells, isolated asdescribed in section 1 above, and the 8-azaguanine-resistant,HAT-sensitive CEM T-lymphocyte line (CEM.8aza^(r/). C), prepared asdescribed in section 2 above. In particular, 7.5×10⁶ cells from Band IIof the Percoll gradient fractionation of patient's mononuclear cellswere subjected to flow microfluorometry and light microscopicexamination. These cells were hybridized to an equal number ofCEM.8aza^(r/). C using polyethylene glycol (mol wt 1000, Sigma, St.Louis, Mo.) as described by Jones, C.M., "T-cell Hybridomas ProducingMacrophage Activation Factors," In: T. Cell Hybridomas, Ed.: M. Taussig,CRC Press., Inc., Boca Raton, Fla., 1985, pp. 56-68, incorporated hereinby reference. The fused cells were cultured for 24 hrs in RPMI 1640 with10% FCS prior to addition of HAT-containing medium (hypoxanthine 1×10⁻⁴M, aminopterin 4×10⁵ M, and thymidine 1.6×10⁻⁵ M). Colonies wereselected after 1 month in HAT media and cloned by limiting dilution.

Individual hybridoma clone colonies which were isolated by thisprocedure were adjusted to a culture density of 5×10⁵ cells/ml andstimulated with 16 ug/ml phytohemagglutinin (PHA; Miles-Yeda, Rehovot,Israel) for 24 hours to stimulate lymphokine production. Thus, hybridomaclones were screened for positive MCF production by mitogen stimulationfollowed by subjecting the resultant hybridoma supernatants to biologicscreening in the human monocyte cytotoxicity assay, which is discussedin detail below.

The human T-cell hybridoma was also successfully maintained in 5% fetalcalf serum (FCS), then grown in serum free media for at least 24 hoursafter stimulation by phytohemogglutinin (PHA). Either tissue culturemedia not supplemented by serum, or a serum-free media described bySachs, L. (1978) Clin. Exp. Immunol. 33:495 and incorporated herein byreference, will support this growth for a limited time span. This cellgrowth procedure permits analysis of MCF prepared in serum-freeconditions.

In particular, supernatants of stimulated and unstimulated clones wereincubated 20 hours with the monocyte monolayer. Serial dilutions of thesupernatant were made to quantitate MCF in each sample. The data in FIG.1 show that all six clones produce a factor(s) which induces humanmonocytes to kill K562. Their phenotypes were studied by FMF and twoclones, FtF3 and FtA5, were uniformly OKT 3+, 4+, and were OKT 8- andOKMT 1-, and IgG-.

Table 1 demonstrates the results obtained when supernatants from tworepresentative MCF-producing hybridomas, FtA5 and FtF3, were subjectedto the in vitro monocyte cytotoxicity assay. Cytotoxicity was measuredby adding a fixed input of supernatant (25, 50 and 100 vl.) to a totaltest volume of 0.2 ml.

                  TABLE I                                                         ______________________________________                                        Specific Cytotoxicity Induced                                                 By Hybridoma Supernatants                                                     Hybrid      Input (ul)                                                                              % Specific Lysis                                        ______________________________________                                        FtA5        25        10.3                                                                50        25.1 ± 6.9                                                       100       38.3 ± 8.4                                           FtF3        25        11.1                                                                50         26.2 ± 10.4                                                     100       37.7 ± 6.8                                           ______________________________________                                    

4. Human Monocyte Cytotoxicity Assay

As noted above, successful practice of the present invention rests onthe ability to successfully identify MCF activity in an in vitro assaywhich has been designed to distinguish MCF activity from the numerousother lymphokine activities produced by T-cells. The assay describedbelow has been derived in part from an assay method reported by Korenand his associates (Fischer et described in the Fischer et al. referencewas found to be al., Cell Immunol., 58:426, 1981). However, the assay asunacceptable in that it is not designed to measure lymphokine inducedcytotoxicity and does not eliminate natural killer cell activity. It,therefore, must be modified as follows.

Human monocyte enriched leukopaks were obtained as a byproduct of theplatelet donor program at M. D. Anderson Hospital, and were preparedwith an IBM Model 2997 Cell Separator. Only healthy volunteer donorswere used. All volunteer donors signed an informed consent, and theprotocol was approved by the Committee for the Protection of HumanSubjects, University of Texas Health Science Center. Mononuclear cellswere prepared by centrifugation over Ficoll/Hypaque. Monocyte monolayerswere prepared by adherence to 96-well flatbottom plates previouslycoated with human serum as described by Golightly et al. (Blood, 61:390,1983, incorporated herein by reference), and allowed to adhere to theserum coated plates for 15-30 mins followed by vigorous washing withwarm (37° C.) Hank's balanced salt solution. This resulted in aconfluent monolayer of >95% esterase positive cells. The monolayer wasincubated overnight to allow for decay of residual natural killeractivity before addition of lymphokine preparations on day 2. Themonolayers were incubated with lymphokine for 20 hrs, washed, and the¹¹¹ In-Ox labeled K562 added as targets.

The ¹¹¹ In-Ox labeled K562 target cells were prepared by the method ofWiltrout et al. (in: Manual of Macrophage Methodology, Ed. H. B.Herscowitz et al., Marcel Dekker, Inc., N.Y., 1981, pp 337-344,incorporated herein by reference). The effector to target ratio was30:1. Spontaneous release averaged 15% (7-20%) in greater than 50experiments. LPS (Lipopolysaccharide) free RPMI I640 (M. A. Bioproducts,Walkersville, Md.) with 10% heat-inactivated AB-negative human serum(FLOW Laboratories, Arlington, Va.) was used throughout the assayprocedure. After 18 hrs incubation of monocyte monolayer with target,the plates were centrifuged and supernatant was removed and cpms werecounted in a Beckman Biogamma 2000. Specific release was calculated asdescribed by Wiltrout, supra. Units of MCF activity were calculated asdescribed by Lohmann-Matthes (Kniep et al. (1981), J. Immunol.,127:417). 20% specific release equals 1 unit of MCF. Other tumor targetsused were HL-60, L5178Y and TU5 (Table V).

The primary distinction between the present assay and the one describedby Fischer et al., is the finding that it is absolutely crucial thatLPS-free media be utilized in order to distinguish the MCF activity frominterfering activities. For the particular activity investigated byFischer et al., the use of LPS-free media was not crucial in that it isnoted by those authors that similar activities were observed regardlessof whether LPS-free media was utilized. However, with respect to MCF,when LPS-containing media is used, a spontaneous release of label occursand induction of lymphokine (MCF) directed cytotoxicity cannot bemeasured.

5. Deposit of Representative Sezary Cell Hybridomas with the AmericanType Culture Collection

By the foregoing procedures, six hybridoma clones were identified whosesupernatants exhibited MCF activity. Two were chosen for furtherphysiochemical and biologic characterization. It has been determinedthat those two hybridomas, designated Ft.A5 and Ft.F3, both secrete thesame MCF biologic profile, as determined by the characterizationcriteria disclosed herein. Accordingly, one of these hybridomas, Ft.F3,has been deposited with the ATCC and accorded ATCC reference numberHB9713.

6. Characterization of MCF A. MCF is Antigenically Distinct FromInterferon Gamma 1) Generation of MCF

MCF was generated by stimulating 5×10⁵, 1×10⁶, 2.5×10⁶, or 5×10⁶ FtF3 orFtA5 cells/ml with 2, 4, 8, 16, or 32 ug/ml PHA (Miles-Yeda, Rehovot,Israel) in RPMI 1640 with 1, 2.5, 5, or 10% FCS (Hyclone, SterileSystems, Logan, Utah) for 24, 48, or 72 hrs. Unstimulated controls weregrown with each to which an equal amount of PHA was added at the end ofincubation. The cells were centrifuged, and the supernatantsfilter-sterilized, and stored at -30° C. For characterization andpurification, MCF was then prepared by stimulating 1×10⁶ cells/ml inRPMI 1640, 1% FCS with 8 ug/ml PHA for 24 hrs, centrifuged, and filtersterilized. This routinely gave approximately 40 U/ml activity.Unstimulated controls to which PHA was added back were used in allexperiments.

MCF was also generated in serum-free media. FtF3 cells were first grownin RPMI 1640 containing 5% FCS. Cells were then washed twice with Hanksbalanced salt solution (HBSS) and once with RPMI 1640. Cells wereresuspended at a concentration of 1×10⁶ /ml or 3×10⁶ /ml in either RPMI1640 containing 10, 5, 1, or 0.1% heat-inactivated, AB-negative humanserum, RPMI 1640 alone, or a serum-free media described by Sachs (Clin.Exp. Immunol., 33:495, 1978, incorporated herein by reference).Phytonemogglutinin (PHA) was added to a final concentration of 0, 0.5,1.0, 2.0 or 4.0 micrograms/ml. Supernatants were collected at 24 and 48hours, filter sterilized, and stored at -70° C. Satisfactory growthcould only be maintained in RPMI 1640 having at least 5% fetal calfserum. However, after FtF3 had been conditioned to grow in media having5% FCS, this cell line was capable of producing MCF under serum-freeconditions as described herein. The total units of MCF recovered werescarcely different among the serum-containing medias or RPMI alone. Theserum-free media of Sachs resulted in a decrease in MCF production of 5to 10 U/ml. In addition, an increase in cell concentration from 1×10⁶cells/ml to 3×10⁶ cells/ml did not increase levels of MCF productionwhich is likely the result of decreased viability at higher celldensity.

2) Interferon Assay

Human interferon activity was measured as inhibition of plaque formationby Sindbis virus on WISH cells as described by Baron et al. (Infect.Immun., 32:449, 1981). Sindbis virus, human alpha, beta, and gammainterferons were prepared by Drs. Samuel Baron and Marlyn Langford,University of Texas Medical Branch, Galveston, Tex. WISH cells wereobtained from Drs. Baron and Langford. Using this plaque inhibitionassay, interferon was not demonstrated in supernatants of FtF3 and FtA5.

3) Treatment of IFN-gamma and MCF With Antisera to Various Interferons

Antibody to a partially purified preparation of native human gammainterferon (Langford et al. (1981), J. Immunol., 126:1620), a 20 peptideN-terminal fragment of recombinant gamma interferon (Johnson et al.(1982), J. Immunol., 129:2357) and human alpha interferon (Langford etal., supra) were prepared as described in the referenced articles. Ininitial experiments, 40 Units of MCF or 100 U IFN-gamma in 1 ml RPMI1640, 1% FCS were incubated with 100 U of each of the antisera abovealone or in combination at 4° C. for 30 min, 1 hr, and 4 hr. Followingthis incubation, serial dilutions of the IFN or MCF were made in RPMI1640, 10%FCS and residual macrophage activating factor activity measuredin the MCF assay.

27 U/ml of partially purified MCF were diluted 1/2 (13.5 U), 1/4 (6.75U), and 1/8 (3.375 U). Serial half-log dilutions were made of thesedilute MCF, and the predicted number of units were confirmed bymeasurement in the MCF bioassay. Next, 27 U/ml of partially purified MCFwere subject to serial half-log dilutions up to a final dilution of 1/8.100 U, 50 U, or 25 U of each of the anti-IFN-gamma, antisera was addedto each of these MCF dilutions and incubated for 1 hr at 25° C. ResidualMCF activity was calculated in the constant antibody titration asrecommended by Kawade (J. Ifn. Res., 4:571-584, 1984). Units areexpressed as described by Lohmann-Matthes (Kniep et al., supra).

Results from two representative sets of experiments are summarized inTable II. Anti-interferon-alpha produced no significant change in %specific release. On the contrary, the presence of this antisera duringthe activation has consistently produced small increases in specificlysis. The antibody to a partially purified preparation of native humaninterferon-gamma (SEA-activated human PBL) produced a decrease of only15% and 10% specific lysis. Antisera to the 20-peptide N-terminalfragment of recombinant interferon-gamma or to a combination ofinterferons alpha and gamma failed to neutralize MCF. The antiserathemselves produced only small changes in spontaneous release of labelfrom the target K562 when added in place of the activating agent(spontaneous release=22.6% with media and monocytes alone; specificrelease=-13.2% with anti=IFN-alpha, =4.66% with anti-IFN-gamma native,and =8.4% with anti-IFN-gamma N-terminus.) More importantly, IFN-gammain amounts up to 1000 U/ml final concentration increased specific lysisonly 6.2%. Because IFN-gamma did not produce significant activation forcytotoxicity in the present assay, direct comparison with MCF could notbe performed.

                  TABLE II                                                        ______________________________________                                        Treatment of MCF with Antisera                                                                 % Specific Lysis                                             Treatment          Exp 1     Exp 2                                            ______________________________________                                        untreated          54.6 ± 3.8                                                                           29.8 ± 5.4                                    anti-IFN-alpha     57.6 ± 5.0                                                                           35.8 ± 4.2                                    anti-IFN-gamma (native)                                                                          40.6 ± 3.8                                                                           19.9 ± 8.5                                    anti-IFN-gamma N-terminus    26.5 ± 8.0                                    anti-IFN-gamma (native) +                                                                        48.8 ± 5.3                                              anti-IFN-alpha                                                                ______________________________________                                    

In order to confirm that increasing concentrations of antibody relativeto MCF units would not affect induction of cytotoxicity, the followingexperiments were performed. Serial checkerboard dilutions of 27 U/mlpartially purified MCF were carried out as described above. In eachexperiment, 100, 50, or 25 U of the anti-IFN-gamma antibodies were addedseparately to MCF dilutions and residual MCF units were measured. Theresults summarized in Table III demonstrate that 100 U anti-IFN-gammanative reduced total bioassayable units by 7 U/ml. This is consistantwith preceding experiments. Addition of 50 U/ml anti-IFN-gamma (native)and all inputs of anti-IFN-gamma N-terminus caused an increase inbioassayable units.

                  TABLE III                                                       ______________________________________                                        Treatment of MCF with Anti-IFN-gamma                                          by the Constant Antibody Technique                                                     MCF (units/ml)                                                                  anti-IFNgamma                                                      Input Ab (U/ml)                                                                          (native)    anti-IFNgamma-N-terminus                               ______________________________________                                         0         27          27                                                     25         28          39                                                     50         58          46                                                     100        20          40                                                     ______________________________________                                    

FtF3 and FtA5 MCF were also capable of activating murine peritonealexudate macrophages for cytotoxicity against the target L5178Y. Thepresence of LPS did not appear to augment cytotoxicity induced by theselymphokines.

B. MCF is Distinct from IL 1, IL 2, TNF and m-CSF 1) Measurement of IL1, IL 2, TNF and CSF Biologic Activity

IL 1 activity was measured using the D10.G4.1. cell line as described byKaye and Janeway (J. Immunol. 133, 2291, 1984). IL 2 activity wasmeasured as described by Bonnard, using cultured human T-cells (Cell.Immunol. 51:390, 1980). TNF activity was measured by direct cytotoxicityagainst L929 cells as described by Gately and Mayer (J. Immunol.116:669, 1976). M-CSF was measured by both murine bone marrow colonyformation (Waheed and Shadduck, Exp. Hematol. (1989) Exp. Hematol.,17:61-65 (Shadduck and Waheed, (1989) Ann. N.Y. Acad. Sci., 554:156-166,and by radioimmunoassay specific for purified human m-CSF.

2) Addition of Lymphokines to MCF Assay

Purified human IL 1 (alpha plus beta), rIL 1a, rIL 1b, (CistronBiotechnology, Pinebrook, N.J., USA), rIL 2 (Genzyme Corp., Boston,Mass., USA), rGM-CSF, or purified m-CSF (Waheed and Shadduck, supra),was added to the human monocyte cytotoxicity assay in order to determinethe ability of lymphokines other than MCF to activate monocytes fortumor cytotoxicity. Antibody to a partially purified preparation ofnative human IFN-gamma, a 20 peptide n-terminal fragment of rIFN-gamma,and human IFN-alpha were used, and neutralization was carried out usingthe constant antibody method as previously described by Jones et al. (J.Immunol. 137:571, 1986). Anti-serum to purified m-CSF, capable ofneutralizing 0, 100 and 1,000 units/ml was added to a preparation ofhuman MCF containing 33 U/ml. These were incubated one hour at roomtemperature. Resultant supernatants were tested in the MCF biologicassay.

Using K562 as a cytotoxicity assay as previously determined,IFN-alpha/beta had no, and IFN-gamma only slight activity. Neither IL 1or IL 2 had any activity in the MCF assay. Anti-sera to m-CSF failed toneutralize MCF activity. The CSF's had effects different from MCF. Theycaused monocyte cell division, altering the effector to target ratio andcausing cells to assume a rounded morphology. MCFs by contrast did notcause cell division, but induced cytotoxicity and caused the cells toassume a macrophage-like morphology. The details of the effects ofcytokines or lymphokines other than MCF, in the MCF assay, are shown inTable IV.

                                      TABLE IV                                    __________________________________________________________________________    Effect of other cytokines in the MCF assay                                    Cytokine                                                                             U/ml % specific lysis                                                                       MCF U/ml                                                                            Specific antisera treatment of                     __________________________________________________________________________                               MCF                                                                                  Exp. 1 Exp. 2                                                          αIFN-αinput                                                              % specific lysis                            IFN-α                                                                          1000 -8.9 ± 8.8                                                                          18.1 ± 2.6                                                   100   -2.1 ± 12.4                                                          10   -5.4 ± 9.2  0  U/ml                                                                              54.6 + 3.8                                                                           29.8 + 5.4                                  1     4.5 ± 3.0  100    57.6 + 5.0                                                                           35.8 + 4.2                                                      αIFN-γ input                                                             Native N-terminus                                                      U/ml   U/ml                                        IFN-γ                                                                          1000  6.2 ± 4.4                                                                          40.0 ± 2.9                                                                       0      27     27                                          100   3.4 ± 3.2  25     28     39                                          10   -3.5 ± 1.1  50     58     46                                          1    -2.2 ± 1.9  100    20     40                                   rIL1α                                                                          10   -0.7 ± 7.6                                                                          24.4 ± 6.5                                                   1    -3.6 ± 5.6                                                            0.1  -4.1 ± 4.1                                                     rIL1β                                                                           10   -1.8 ± 9.6                                                                          24.4 + 6.5                                                      1    -4.9 ± 4.6                                                            0.1  -8.7 ± 2.8                                                     pIL1(α + β)                                                               100  -10.2 ± 4.0                                                                         18.1 ± 2.6                                                   10   -5.2 ± 2.7                                                            1    -5.4 ± 7.9                                                            0.1  -10.9 ± 3.3                                                    rIL2   1000  2.7 ± 6.2                                                                          20.0 ± 2.0                                                   100  -3.2 ± 3.5                                                            10   -4.6 ± 4.3                                                            1    -10.2 ± 17.7                                                   GM-CSF 200  -0.6 ± 4.9                                                                          20.0 ± 4.4                                                   100   5.7 ± 3.1                                                            50    -2.5 ± 15.9                                                                              am-CSF input                                                                         MCF activity                                                           U/ml   U/ml                                        m-CSF  1730 -3.9 ± 4.7                                                                          50.0 ± 3.9                                                                       0      33.3                                               173   14.5 ± 4.6 100    40.0                                               17.3  26.0 ± 7.5 1000   50.0                                        __________________________________________________________________________     Various cytokines were added to the MCF bioassay. Specific antisera were      added to MCFcontaining supernatants to determine whether any MCF activity     could be neutralized by antisera to other cytokines.                     

3) MCF is Distinct from Natural Killer Cells (NK)

In order to remove human NK cells from whole human peripheral bloodmononuclear cells, anti-Leu-11B was used (Becton Dickinson, MountainView, Calif., USA) (Itoh et al., J. Immunol. 134:802, 1985). Humanmonocyte monolayers were treated with anti-Leu-11B and subjected toeither activation with MCF for measurement of cytotoxicity, or stainingwith trypan blue for viability. Human monocyte monolayers treated withanti-Leu-11B plus complement and activated with MCF did not diminish MCFmediated cytolysis, nor was viability at the monocyte monolayersdecreased when compared to control monolayers. Monocyte monolayers wereactivated with either crude MCF (23.8 U/ml) or MCF prepared by MatrexGel Red A chromatography (83.3 U/ml). Cytotoxicity was measured usingK562, HL60, L5178Y or TU5. Specific release of MCF was comparable withboth NK-sensitive and NK-resistant (TU5 and L5178Y) cells. (Table V)

                  TABLE V                                                         ______________________________________                                        MCF-induced cytotoxicity against tumor targets                                Target            MCF (units/ml)                                              ______________________________________                                        crude MCF:                                                                    K562              23.8 ± 9.1                                               HL-60             23.5 ± 1.5                                               partially purified MCF:                                                       K562               83.3 ± 12.3                                             L5178Y            78.4 ± 8.4                                               TU5               100.5 ± 26.8                                             ______________________________________                                         Monocyte monolayers were activated with either crude MCF or MCF prepared      over Matrex Gel Red A for 24 h before the addition of targets.                Units were calculated as the reciprocal of the dilution giving 20%            specific lysis.                                                               These are the results of 3 experiments run in quadruplicate.             

TNF could not be demonstrated in supernatants collected from humanmonocytes incubated with 20 U/ml MCF for 20 h, washed and incubated for24 h (dilution of 1:10). However, IL 1 was present at concentrations ofup to 100 U/ml in MCF-activated monocyte supernatant.

C. Response of MCF to Enzyme Treatment

In order to treat MCF supernatants with enzymes, 10 ml of thesupernatant (28.5 units/ml) were treated with 1 mg/ml trypsin at pH 7.4,1 mg/ml chymotrypsin at pH 7.4, 0.5 mg/ml DNase at pH 7.4, or 40units/ml RNase at pH 5 for 1 hour at 25° C. The result of treating MCFwith chymotrypsin was that there was a reduction in the biologicalactivity of MCF by 54.4%. Trypsin, RNAse and DNAse had no significanteffect. The experiments were repeated using insolubilized enzymes tominimize the possibility that enzymes could be carried over into thebioassay and could explain the results. However, the results wereentirely comparable to those using soluble enzymes. Chymotrypsin reducedactivity from 28.5 U/ml to 12.5 units, whereas trypsin RNAse and DNasetreatment showed no significant differences after one hour of treatment,as measured by bioassay for MCF and determined by students T test.

D. Effects of Tunicamycin and Other Agents on MCF

Urea treatment was accomplished by adding 3 grams of solid urea to 10 mlMCF (28.5 U/ml) (5M urea final). Extraction of MCF withbutanol-diisopropyl ether was performed. FtF3 cells Were cultured with0, 1, 2.5 or 5.0 ug/ml tunicamycin. Urea decreased biological activityin the MCF containing supernatants by 35.7%. MCF supernatants wereextracted with butanol-diisopropyl ether; the aqueous phase contained36.7% less MCF than the control preparation. Biosynthesis in thepresence of either 2-mercaptoethanol or tunicamycin resulted in nosignificant change in biological activity of MCF.

E. Effects of Metabolic Inhibitors on MCF

FtF3 cells at a concentration of 1×10⁶ /ml in RPMI 1640/10% FCS wereincubated 4 hours with either actinomycin D, cycloheximide, or puromycin(all at 0 to 100 ug/ml, Sigma). Tests for viability after treatment wereperformed by trypan blue exclusion and supernatants were collected bycentrifugation. In a second set of experiments FtF3 cells at the sameconcentration but using 0.1% human serum were incubated for 5 hours witheither cycloheximide or puromycin. Uptake experiments were performed byadjusting FtF3 cells to 1×10⁶ /ml in media after treatment withinhibitor, and adding either 5,6-³ H-uridine (ICN Radiochemicals,Irvine, Calif., USA, 49,Ci/mmol) to actinomycin D treated cells or ³ HL-amino acid mixture (25.5 mCi/mg) to cycloheximide-treated cells at 1uCi to 1×10⁵ cells. Cells were subsequently lysed and counted by liquidscintillation.

When MCF was grown in the presence of the metabolic inhibitors,actinomycin D totally suppressed production of MCF in a dose-dependentmanner accompanied by a corresponding fall in ³ H-uridine uptake. (FIG.5) Cycloheximide suppressed but did not abolish MCF production. (FIG. 6)At intermediate doses (25 and 50 ug/ml) some escape from suppression wasnoted. Puromycin, like cycloheximide, suppressed but did not totallyabolish MCF production under these conditions. (FIG. 7) A correspondingpattern was observed using tritiated amino acids and examining thepattern of incorporation in TCA insoluble material.

Actinomycin D had no effect, and cycloheximide had only a slightstimulatory effect, on MCF production of non-stimulated (non-PHAtreated) cells. Puromycin at doses of 10-50 ug/ml appeared to stimulateproduction of MCF by FtF3 cells not activated by lectin. Cycloheximideand puromycin therefore provide reversible inhibition. When inhibitorwas present for the entire incubation period, lectin-induced MCFproduction is not suppressed by cycloheximide but puromycin wassuppressive in a dose-dependent manner.

F. MCF Behavior in Other Bioassays

The biological activity of MCF was checked in other bioassays to probethe issue of multiple biologic activities. Supernatants from FtF3containing 25 U/ml of MCF activity were substituted for IL 1 and IL 2 intheir respective bioassays. MCF demonstrated no IL 1 or IL 2 activity.MCF had no TNF, m-CSF, or IFN-like antiviral biological activity whencompared to rTNF, purified human m-CSF, IFN-gamma or alpha/betarespectively.

G. Physicochemical Characterization of MCF 1) pH Stability

pH stability of MCF was compared to IFN-gamma by dialyzing 40 U MCF or100 U IFN-gamma in Spectropor tubing (molecular weight cutoff of 5×10³Daltons) against 0.1M glycine-HCI, pH 2.0, 0.1M TRIS-HCl, pH 5.0, 0.15MPBS, pH 7.4, 0.15M PBS, pH 8.0, or 0.1M TRIS, pH 10.0 for 4 hrs at 25°C. The samples were then dialyzed against Dulbecco's PBS, pH 7.4, torestore neutrality prior to bioassay. IFN-gamma was found to be totallyinactivated at pH 2. However, as demonstrated by Table VI, MCF was foundto be stable at pH 2. However, partial inactivation of MCF hasconsistently been noted at pH 8.0.

                  TABLE VI                                                        ______________________________________                                        pH Stability of MCF                                                                  pH   % Specific Lysis                                                  ______________________________________                                               2    41.7 ± 8.6                                                            5    41.1 ± 6.4                                                            7.4  36.8 ± 3.2                                                            8    19.6 ± 4.6                                                            10   45.4 ± 5.4                                                     ______________________________________                                    

2) Heat Stability of MCF

To test heat stability of MCF relative to IFN-gamma, 40 units of MCF and100 U IFN-gamma in 1 ml RPMI 1640/1% FCS were heated for 2 hrs in aconstant temperature bath to study heat denaturization. As demonstratedby Table VII, MCF was stable at temperatures up 60° C., but was totallyinactivated at 100° C. IFN-gamma was not stable at temperatures higherthan 4° C. for periods of 2 hrs or longer.

                  TABLE VII                                                       ______________________________________                                        Heat Stability of MCF                                                         Temperature   % Specific Lysis                                                ______________________________________                                         4°    53.3 + 4.3                                                      37°    48.6 + 4.9                                                      60°    48.0 + 3.4                                                      100°   0.0                                                             ______________________________________                                    

7. Purification of MCF A. Gel Filtration of MCF

Gel filtration experiments were performed both to determine anapproximate molecular weight and to begin preparation of a substantiallypurified fraction. 180 ml of MCF from PHA stimulated FtF3 supernatant(7.2×10³ U MCF) was concentrated 20-fold by pressure dialysis over anAmicon YM-10 membrane, applied to a 2.5×60 cm. column of Bio-Gel P100,equilibrated with PBS and eluted at a flow rate of 1.5 ml/min. 7.5 mlfractions were collected and were assayed undiluted, 1/2 and 1/5 for MCFactivity. The column was calibrated with aldolase (158K), ovalbumin(45K), chymotrypsin (25K), and ribonuclease A (13.7K) (Pharmacia).

The data in FIG. 2 demonstrated that two peaks of biologic activity wereobtained. The first peak co-eluted with the major protein present,bovine serum albumin (from the fetal calf serum present in the culturemedia). The second peak of monocyte cytotoxicity inducing activityeluted in a region with an apparent molecular weight of approximately11,500 Daltons.

B. Precipitation with Ammonium Sulfate

Initially, ammonium sulfate precipitation was attempted as a method forpurification and concentration. MCF appeared to precipitate in the30-50% range but resulted in greater than an 85% loss in biologicactivity. Therefore, ammonium sulfate precipitation was not pursuedfurther as a means for purification.

C. Binding of MCF to Matrex Gel Resins

Two ml of each of the Matrex Gel Resins (Amicon) was washed with 5M Ureaand then washed with 20 ml PBS in 1.5M NaCl, pH 7.4. Four ml of FtF3supernatant (160 U MCF in RPMI 1640, 1%FCS) was passed twice over eachof the Matrex Gel Resins (Blue A, Red A, Orange A, Green A, and Blue B),washed with 2 volumes of starting buffer, and eluted stepwise with 1volume each 0.5M NaCl. 1.0M NaCl, and 2 volumes 1.0M NaCl/ 50% ethyleneglycol. The starting material, wash, and each fraction was then dialyzedat 4° C. for 24 hrs (Spectrapor tubing, 5×10³ D molecular weight cutoff) against 3 changes of PBS, pH 7.4, and then 4 hrs against distilledH₂ O. The samples were placed in a glass tray and covered with SephadexG-10 to reduce volume to approximately 1 ml. Each sample was thenassayed for units MCF activity.

MCF was bound and could not be eluted from Matrex Gels Orange A, GreenA, and Blue B. However as summarized in FIG. 3, MCF bound to both BlueA, and Red A. Red A bound 31.3% of the starting material and eluted with1.0M NaCl. Blue A bound only 10.5% of the starting and was not studiedfurther. 175 ml of FtF3 supernatant/RPMI 1640, 1%FCS containing 7×10³units MCF was passed twice over a 2.5×30 cm column Matrex Gel Red A. Thecolumn was washed with two column volumes of starting buffer and elutedin a single step with 1.0M NaCl. The peak activity was present infractions 11-14. Two×10³ units in 20 ml (1.45 mg protein/ml) werecollected. This preparative procedure resulted in a 2.5 foldconcentration and gave a 28.5% yield. No IFN-gamma was detected in thispreparation.

D. Polyacrylamide Gel Electrophoresis (SDS-PAGE)

To determine the molecular weight of MCF by an alternate procedure,samples having MCF activity were subjected to SDS-PAGE. In particular,200 ul (20 U MCF/ 290 ug protein) prepared by chromatography over MatrexGel Red A was mixed 1:1 with sample preparation buffer (0.0625 mlTRIS-HCl, 2% SDS, 5% 2ME, 10% sucrose, and 0.002% bromphenol blue),heated 3 min at 100° C., and centrifuged at high speed (1 min in anAdams microfuge). 100 ul aliquots were loaded into each of 4 lanes of a1.5 mm thick SDS slab gel (BRL vertical gel apparatus, Gaithersburg,Md.) using a 10% polyacrylamide gel, prepared according to Laemmli(Nature, 227:680, 1970), and run at 90 V through the stacking gel and200 V through the running gel. The gel was cut into 1 cm slices,crushed, and eluted with 2 washes of 5 ml of PBS/0.1% SDS pH 7.4 , for12 hrs at 4° C. The samples were placed in a glass dish, and coveredwith Sephadex G-10 to reduce sample volume to 1.5 ml. Pharmaciastandards prepared in the same buffer and run in companion lanes wereused to determine molecular weight; phosphorylase B (94K), bovine serumalbumin (67K), ovalbumin (43K), carbonic anhydrase (30K), soybeantrypsin inhibitor (20K), and alpha-lactalbumin (14.4K). A companion gelwas divided and stained with 1) Coomasie Brilliant Blue and 2) by asilver nitrate method (Oakley et al., Anal. Biochem., 105:361, 1980).

Two peaks of MCF activity were obtained, one with a molecular weightbetween 78,400 and 63,600 Daltons which was associated with a majorprotein band, bovine serum albumin (specific activity 610 U MCF/mgprotein), and a second peak between 18,100 and 14,700 (specific activity1350 U MCF/mg protein) (FIG. 4). Using a silver stain method, a proteinband was detected associated with the major peak of MCF activity at14,700 Daltons, which could not be detected using Coomassie BrilliantBlue. Recovery of biologic activity from the gel wa greater than 90%.

To determine the molecular weights of MCF prepared from the supernatantsof cells grown in serum-free media, similar procedures were followedwith some exceptions, e.g., 15% SDS-PAGE, and the following lowmolecular weight standards: ovalbumin (43K), α-chymotrypsinogen (25.7K),a-lactaglobulin (18.4K), lysozyme (14.3K), bovine trypsin inhibitor(6.2K), and insulin A or B chain, (2.3K and 3.4K). One half of the gelwas sliced, crushed and eluted as previously described to determinebiologic activity. A corresponding gel was washed briefly in distilledwater and placed against 2 sheets of nitro-cellulose (Bio Rad, Richmond,Calif.) as described by Palade (Gershoni and Palade, Anal. Biochem.124:396,1982); electroluted 4 hr at 200 mA using a Bio RadTransblotapparatus in a Tris Glycine buffer, and stained with colloidalgold Aurodye (Anal. Biochem. 145:315, 1985). (FIGS. 8A and 8B). Underserum-free conditions, the higher molcular weight species is found at 29kilodaltons.

Treatment Protocols

Due to precautions which are necessarily attendant to every newpharmaceutical, due both to consideration of patient safety and federalnew drug regulations, the MCF of the present invention has not beentested as yet in a clinical setting in human subjects. However, the invitro activity of MCF in stimulating monocytes to kill tumor cells,along with the recent clinical success of interleukin II, is believed todemonstrate the utility of the present invention in this regard. Thefollowing embodiments are therefore prophetic and represent the bestmode contemplated by the present inventor of carrying out the practiceof the invention in various clinical settings.

1. Antitumor Therapy A. Direct Infusion

It is believed that MCF will prove to be useful in the treatment ofvarious tumors, and in particular, tumors of the blood forming organssuch as leukemias, or solid tumors which have been described asinfiltrated by machrophages, by way of direct intravenous infusion ofpharmaceutical compositions which include MCF. Such compositions wouldinclude effective doses of either MCF alone, or in combination withother therapeutic agents such as interleukin II, interferon, tumornecrosis factor or cytoxan. Interleukin II may be obtained as disclosedby numerous U.S. patents, including for example, U.S. Pat. Nos.4,407,945 and 4,401,756, incorporated herein by reference. Cytoxan(cyclophosphamide) is a commercially available antineoplastic agent.Interferon is also commercially available as disclosed herein, and itsclinical use has been reviewed and described in detail in numerouspublications, including, for example, in Goldstein et al. (1986), Can.Res., 46:4325-4329, incorporated herein by reference. Moreover,Goldstein discloses in detail the suggested and reported dose regimensfor interferon antitumor therapy. Preparation of Tumor Necrosis Factorand its use is known in the art as exemplified by U.S. Pat. Nos.4,457,916; 4,529,594; and 4,447,355 and as further disclosed by Carswellet al. (1975), Proc. Natl. Acad. Sci, USA, 72: 3666; Ruff et al. (1980),J. Immunol., 125: 1671; Matthews et al. (1980), Br. J. Cancer, 42: 416;and Lu et al. (1986), Cancer Res., 46(9): 4357, all of the foregoingreferences being incorporated by reference. Therefore, it is consideredthat use and dosages of MCF treatment, alone or in combination withthese agents, is well within the skill of the art in light of thepresent specification.

MCF could be given daily by continuous infusion or given on alternativedays with interleukin-2 or interferon being given on the other day. Sucha treatment would be possible since the cytotoxic effect of MCF seems tolast for about approximately 24 hours. Alternatively a large initialdose of Cytoxan could be given which should deplete suppressorT-lymphocytes followed by continuous infusion of MCF. Doses of MCF Wouldof course have to be determined by experimental methods which are wellknown to skilled immunologists. However, dosages will likely be at leastan order of magnitude lower than dosages of interferon gamma.Interferons are usually given as an IM dose of 3 million units thriceweekly although one would have to take into account whether total bodywater is being saturated. With a new agent of any type one would have toinitiate a phase I trial first to establish levels at which unacceptabletoxicity is reached.

B. Adoptive Immunotherapy

Adoptive immunotherapy is a new approach to treating metastatic cancerin which immune cells with antitumor reactivity are transferred to thetumor-bearing patient. Much of this work has been pioneered by Dr.Steven Rosenberg and is discussed in more detail in Resenberg et al.(1977), Adv. Cancer Res., 25:323 and Rosenberg (1984), Cancer Treat.Rep., 68:233, both incorporated by reference. In particular, interleukinII, also referred to as T-cell growth factor, has been shown to be auseful adjuvant to adoptive immunotherapy, wherein it is used tostimulate killer T-cell development (see, e.g., Rosenberg (1985), J.Natl. Cancer Inst., 75:595, incorporated herein by reference). Moreover,adoptive therapy utilizing interleukin II has demonstrated applicabilityin the treatment of a variety of advanced metastatic cancers in humans(Rosenberg et al. (1985), N. Eng. J. Med., 313:1485, incorporated hereinby reference).

Accordingly, it is submitted that the MCF of the present invention canbe utilized in an adoptive immuno-therapy protocol in a manner similarto interleukin II. In particular, it is believed that the followingproposed protocol will serve as a sufficient basis to teach thoseskilled in the art of adoptive immunotherapy to utilize MCF in thismanner.

Monocytes will be harvested by cell separation using, for example, anIBM cell separator using accepted techniques. These cells would then beincubated with approximately 4 units/ml of MCF overnight followed byslow continuous infusion of the induced cells into the patients. Suchtherapy could initially be given 2 to 3 times a week. However, becauseof the long life span of monocytes it could perhaps be given at moreinfrequent intervals stretched over a much longer period to time toinsure that infiltration into the tumor occurs.

C. Diagnostic Utility

MCF will additionally be of value as a clinical diagnostic aid. Forexample, antibodies having specificity for MCF will provide an abilityto determine MCF blood levels, thereby assisting in maintainingtherapeutic blood levels and perhaps in the diagnosis of T-cellmalignancies which may be accompanied by high serum MCF levels.Moreover, MCF antiserum may be of use in evaluating T-cell function innormal individuals. Similar uses have been described for the so-calledmelanoma-associated antigen.

The development of antibodies to a particular antigen whether polyclonalor monoclonal, are well known in the art and can readily be achieved byskilled immunologists. This is the case even where the particularmolecule is not antigenic in and of itself, through either theattachment of an immunostimulating ligand such as keyhole limpethaemocyanin, or by finding a species wherein the molecule is antigenic.

In the case of MCF it is believed that an antibody can readily bedeveloped by either a polyclonal or monoclonal approach. For example,rabbits should be immunized with 100 micrograms of MCF in completeFreund's adjuvant into each of four sites and later boosted with MCF inincomplete Freund's adjuvant. Once a heteroantisera has been developedand quantitation determined by immunodot assay, western blotting, ELISAand various other immunodiagnostic techniques may be performed with sucha hetero-antiserum. Monoclonal antibodies may be developed by a numberof accepted techniques, for example, as disclosed by U.S. Patent Nos.4,172,124 and 4,271,145, both to Koprowski et al., incorporated hereinby reference.

For in vitro diagnostic work, for example, in an immunoassay toquantitate serum MCF levels, the MCF antibody will be used mostpreferably in an ELISA assay which employs the antibody together with animmuno detection reagent capable of detecting quantitatively specificimmune complex formation.

However, in general, immunodiagnostic kits would include reagentsappropriate for either detecting patient-generated anti-MCF antibodies(e.g. circulating antibodies) or detecting MCF, for example,tumor-generated MCF, in biologic fluids or tissues from patients. Asused herein, a biologic fluid or tissue includes any fluid or tissueobtained from a patient, including, for example, urine, serum, plasma,and biopsy samples. In the case of MCF antibody-detection kits, suchkits would include antigenically pure, and preferably titrated, MCFtogether with an immunodetection reagent. By antigenically pure MCF ismeant an MCF preparation which does not substantially cross-react withnon-MCF directed antibodies. Sufficient antigenic purification could beachieved through immunopurification by adsorption with normal sera orchromatography over anti-MCF antibodies as is known in the art.

In the case of MCF antigen detection kits, such kits would typicallyinclude antigenically pure, preferably titrated, anti-MCF antibody. Byantigenically pure is meant antibody which will not substantiallycross-react with antigens other than MCF. As with MCF antigenpurification, polyclonal antibody purification could be achieved byimmuno chromatography. However, a preferred antibody would be amonoclonal antibody.

In either case, immunodetection kits would include an immunodetectionreagent for detecting and/or quantifying the occurrence of specificimmunoreactions involving MCF. Typically such reagents include, forexample, a radioactive or enzyme-linked ligand. Such ligands aretypically associated with either the antibody, antigen or a secondantigen or antibody. As noted, a preferred immunodetection system arethe various systems based on the ELISA assay. For a further descriptionof the ELISA assay and the various immunodetection reagents, pleaserefer to U.S. Pat. Nos. 4,454,233 and 4,446,232, both incorporatedherein by reference. It is believed that these patents providesufficient disclosure to enable the use of antibody to MCF in a clinicalimmunoassay.

The present invention has been disclosed in terms of specificembodiments which are believed by the inventor to be the best modes forcarrying out the invention. However, in light of the disclosure herebyprovided, those of skill in the various arts will recognize thatmodifications can be made without departing from the intended scope ofthe invention. For example, although the present invention is disclosedin terms of a Sezary cell hybridoma for MCF production, it is clear thatother types of T-cells may be employed. Additionally, numerousembodiments are likely possible for isolation of the factor. Moreover,as biological characterization of the factor progresses, it is likelythat more refined and simpler assays will be developed for MCFidentification. For example, once an antibody to MCF has been developed,such antibody can be used directly to assay for MCF production by thevarious cell populations. These and all other modifications andembodiments are intended to be within the spirit and scope of thepresent invention and appended claims.

What is claimed is:
 1. A method of treating cancer in a patientcomprising infusing the patient with a pharmaceutical compositioncomprising a substantially purified monocyte cytotoxicity inducingfactor characterized by the following properties:(a) capability ofinducing human monocytes to a cytotoxic state; (b) retention ofbiological activity following treatment at pH 2 for one hour; (c)retention of biological activity following treatment at 60° C. for onehour; (d) ability to bind to Matrex Gel Red A under low-salt conditionsand elute from Matrex Gel Red A under high-salt conditions; and (e)retention of biological activity in the presence of anti-serum tointerferon gamma, interferon alpha, or a combination of anti-sera tointerferon alpha and gamma.
 2. The method of claim 1, wherein the factorexhibits an apparent molecular weight of about 29 kilodaltons uponSDS-polyacrylamide gel electrophoresis after production under serum-freeconditions.
 3. The method of claim 1, wherein the factor exhibits anapparent molecular weight of between about 14.7 and 18.1 kilodaltonsupon SDS-polyacrylamide gel electrophoresis.
 4. A method of treatingcancer in a patient comprising the steps of:(a) obtaining humanmonocytes; (b) incubating the monocytes with an amount of a humanmonocyte cytotoxicity inducing factor effective to activate monocytecytotoxicity, said monocyte cytotoxicity inducing factor characterizedby the following properties:i) capability of inducing human monocytes toa cytotoxic state; ii) retention of biological activity followingtreatment at pH 2 for one hour; iii) retention of biological activityfollowing treatment at 60° C. for one hour; iv) ability to bind toMatrex Gel Red A under low-salt conditions and elute from Matrex Gel RedA under high-salt conditions; and v) retention of biological activity inthe presence of anti-serum to interferon gamma, interferon alpha, or acombination of anti-sera to interferon alpha and gamma; and c) infusingthe patient with the activated monocytes.
 5. The method of claim 4,wherein the factor exhibits an apparent molecular weight of about 29kilodaltons upon SDS-polyacrylamide gel electrophoresis after productionunder serum-free conditions.
 6. The method of claim 4, wherein thefactor exhibits an apparent molecular weight of between about 14.7 and18.1 kilodaltons upon SDS-polyacrylamide gel electrophoresis.
 7. Themethod of claim 4, wherein the monocytes obtained in step (a) are fromthe patient treated in step (c).
 8. The method of claim 4, wherein themonocytes are harvested by cell separation.
 9. The method of claim 4,wherein the monocytes are incubated with about 4 units of monocytecytotoxicity inducing factor per milliliter of monocytes.